Direct administration of mesenchymal stem cell‐derived mitochondria improves cardiac function after infarction via ameliorating endothelial senescence

Abstract Mitochondrial dysfunction is considered to be a key contributor to the development of heart failure. Replacing injured mitochondria with healthy mitochondria to restore mitochondrial bioenergy in myocardium holds great promise for cardioprotection after infarction. This study aimed to investigate whether direct transplantation of exogenous mitochondria derived from mesenchymal stem cells (MSC‐mt) is beneficial and superior in protecting cardiac function in a mouse model of myocardial infarction (MI) compared to mitochondria derived from skin fibroblast (FB‐mt) and to explore the underlying mechanisms from their effects on the endothelial cells. The isolated MSC‐mt presented intact mitochondrial morphology and activity, as determined by electron microscopy, JC‐1 mitochondrial membrane potential assay, and seahorse assay. Direct injection of MSC‐mt into the peri‐infarct region in a mouse MI model enhanced blood vessel density, inhibited cardiac remodeling and apoptosis, thus improving heart function compared with FB‐mt group. The injected MSC‐mt can be tracked in the endothelial cells. In vitro, the fluorescence signal of MSC‐mt can be detected in human umbilical vein endothelial cells (HUVECs) by confocal microscopy and flow cytometry after coculture. Compared to FB‐mt, MSC‐mt more effectively protected the HUVECs from oxidative stress‐induced apoptosis and reduced mitochondrial production of reactive oxygen species. MSC‐mt presented superior capacity in inducing tube formation, enhancing SCF secretion, ATP content and cell proliferation in HUVECs compared to FB‐mt. Mechanistically, MSC‐mt administration alleviated oxidative stress‐induced endothelial senescence via activation of ERK pathway. These findings suggest that using MSCs as sources of mitochondria is feasible and that proangiogenesis could be the mechanism by which MSC‐mt transplantation attenuates MI. MSC‐mt transplantation might serve as a new therapeutic strategy for treating MI.


| INTRODUCTION
Ischemic heart diseases remain the leading cause of death worldwide.
Mitochondria are the power plant of the cardiomyocyte, generating more than 95% of the cardiac ATP. Complex cellular responses to myocardial infarction (MI) converge on mitochondrial malfunction, which persists and increases after ischemia, determining the extent of cellular viability and postischemic functional recovery. In a quest to ameliorate various points in the pathway from mitochondrial damage to myocardial necrosis, exhaustive pharmacologic and genetic tools have targeted various mediators of ischemia and have been used in procedural techniques without applicable success. An alternative therapeutic intervention to reverse dysfunction and restore cell normality is to transplant healthy mitochondria into the ischemic heart. The functional exogenous mitochondria will replace the harmed ones, ensuing cardioprotective functions. [1][2][3] Mitochondria transplantation opens a novel horizon for treating MI. Injecting isolated viable respiration-competent mitochondria into the ischemic zone just before reperfusion would reverse postischemic functional deterioration and suppress cellular apoptosis and limit infarct size. 4,5 The first-in-man pilot clinical application of autologous rectus abdominis muscle mitochondrial transplantation in pediatric patients suffering from myocardial ischemia-reperfusion injury resulted in improvement in myocardial function. 2,6 However, current mitochondria sources come mainly from autologous somatic cells or tissue, such as skeletal muscle, 7 cardiac muscle, 4 or pectoralis major muscle, 5 which make harvesting mitochondria an invasive procedure for the donor and potentially raise an ethical problem that might limit clinical application of the procedure. Thus, there is an urgent need to find an alternative source of mitochondria. Damaged mitochondria and byproducts such as damageassociated molecular patterns (DAMPs) are released as stress signals during cellular injury. MSCs signal these factors and elevated reactive oxygen species (ROS) levels, and enhance their bioenergetics and initiate mitochondria donation to the injured recipient cells. 11 In a coculture system of MSCs with epithelial cells, MSCs showed a high capacity to donate their mitochondria via cytoplasmic bridges and reversed rotenone-induced epithelial cell injuries, while smooth muscle cells and fibroblasts showed less capacity for delivering mitochondria, leading to relatively fewer therapeutic effects. 12 In a mouse model of LPS-induced acute lung injury, instilled MSCs formed gap junction channels within the alveolar epithelium and released mitochondria-containing MVs that increased alveolar ATP concentrations, while fibroblasts failed to form gap junctions and rescue the cell bioenergetics in vivo. 13 These data indicate that MSCs have the potential to serve as a source of mitochondria. However, no attempt has been made to assess the potential protective effects of MSC mitochondria against MI.
Here, we investigated whether direct transplantation of isolated MSC mitochondria is applicable and beneficial in a mouse model of MI, and we compared the therapeutic efficacy of MSC mitochondria  F I G U R E 2 Legend on next page.
(FSC intensity) in freshly isolated MSC-mt and stock MSC-mt under normal conditions and after CCCP treatment ( Figure S1B). ADP, a potent regulator of mitochondrial ATP synthesis, stimulates State III respiration. FCCP disrupts the synthesis of ATP by transporting protons across mitochondrial inner membrane. Freshly isolated MSC-mt and stock MSC-mt showed responses to the ADP and FCCP, as determined by the seahorse assay, suggesting MSC-mt were bioenergetic active ( Figure 1e). In general, we confirmed that the morphological and functional characteristics of the MSC-mt, and MSC-mt can be temporarily stored at À80 C for at least 14 days and still retained mitochondria function.  HUVECs (cell trace green labeled) (Videos S1 and S2).

| MSC-mt protected HUVECs from oxidative stress
To determine whether the internalized MSC-mt exerted cytoprotective effects and to compare the effectiveness of MSC-mt and FB-mt, F I G U R E 2 Transplantation of mesenchymal stem cells mitochondria (MSC-mt) improved heart function following infarction. (ai) Representative images of echocardiography taken at 28 days after myocardial infarction (MI) and (aii) measurements of ejection fraction (EF) and fraction shortening (FS). n = 8. ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001 by a one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (bi) Representative images of Masson's trichrome staining and (bii) results from the quantitative analysis of infarction size and infarct wall thickness. n = 8. *p < 0.05, **p < 0.01, and ***p < 0.001 by a one-way ANOVA followed by Bonferroni post hoc test. (ci) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and (cii) results from the quantitative analysis of apoptosis at 28 days after MI. n = 4. *p < 0.05, ***p < 0.001 by a one-way ANOVA followed by Bonferroni post hoc test. (di) Representative images and (dii) results from the quantitative analysis of CD31 and α-SMA density in the border zone of ischemic hearts. n = 4. *p < 0.05, **p < 0.01, and ***p < 0.001 by a oneway ANOVA followed by Bonferroni post hoc test. (ei) Representative immunostaining and (eii) flow cytometry analysis of mitotracker red in the ischemic heart at 7 days post-MI. n = 4. **p < 0.01 by an unpaired t-test. (f) Measurement of ATP content in infarcted heart. n = 5. *p < 0.05, **p < 0.01, and ***p < 0.001 by a one-way ANOVA followed by Bonferroni post hoc test.

| Exogeneous mitochondria enhanced angiogenesis
As we observed an enhanced angiogenesis after MSC-mt transplantation, we next examined whether exogeneous mitochondria regulates angiogenic processes. HUVECs were cocultured with FB-mt or MSCmt for 4 h, and tube formation was analyzed. Marked enhancement of tube formation was observed in the FB-mt and MSC-mt-treated groups, especially in the MSC-mt-treated HUVECs, as measured by increased branch points and total tube length ( Figure 5a). An in vivo

| MSC-mt ameliorated oxidative stress induced endothelial senescence via ERK pathway
The accumulation of senescent endothelial cells impairs the function of angiogenesis and myogenesis after MI, leading to ventricular remodeling and heart function deterioration. 17 We studied whether MSCmt treatment affected endothelial senescence at 7 days post-MI.
While there was no detectable P21 signal in the sham group (data not shown), the ratio of senescent marker P21 to endothelial marker

| DISCUSSION
The current study offers several major findings ( Figure 7). First, isolated MSC-mt presented intact membrane structure, Δψm and bioenergetics, all of which will be critical for subsequent functional applications. Next, MSC-mt administration attenuated myocardial infarct size, increased angiogenesis and improved cardiac performance following infarction, and the protection effects was superior to FB-mt.
Third, MSC-mt could be incorporated into recipient ECs, where they protected the ECs from apoptosis, reduced mitochondrial superoxide production, increased ATP content levels, and promoted proliferation and angiogenesis. Forth, MSC-mt inhibited oxidative stress induced EC senescence via ERK pathway.
Proper mitochondrial function is necessary in the heart, which is of high-energy demand. Functional abnormalities of cardiac mitochondria in MI lead to enhanced oxidative stress, diminished ATP production and energy supply and increased cell apoptosis. 19 In 1982, internalization of exogenous mitochondria was reported by simple coincubation in vitro, without transfection reagents, medium supplements or any other interventions. 8  derived from mature cells. In a coculture study of MSCs with epithelial cells, mitochondria donated by MSCs effectively attenuated rotenone-induced ROS production in epithelial mitochondria, whereas fibroblasts or smooth muscle cells were unable to deliver cytoprotection. 12 In a mouse acute lung injury model, MSCs transferred mitochondria to the alveolar epithelium and thus increased alveolar ATP, whereas fibroblasts failed to donate mitochondria or rescue ATP production. 13 The concept of MSC-derived mitochondria organelle transplantation has been proposed as a revolutionary strategy for the treatment of MI, 22 but there has been no attempt to test this proposed MI therapy to date.
The current study provides fundamental evidence for the applica- and increased tube formation in brain endothelial cells. 27 We showed an increased blood vessel density in the ischemic hearts in the MSCmt-treated mouse group compared with that of the control group, and the improvement was superior to FB-mt (Figure 2d). These results were consistent with the in vitro study, in which MSC-mt treatment remarkably enhanced tube formation in HUVECs (Figure 5a Endothelial cells comprise $60% of the noncardiomyocytes in the heart. 28 Endothelial cell senescence can be triggered by oxidative stress or vascular inflammation. 29 The accumulation of senescent endothelial cells induced angiogenesis dysfunction, impairing repair after MI. 30 Therapeutically, targeting senescent endothelial cell populations remains an attractive strategy for the treatment of cardiovascular diseases. Activating the antiaging protein Nrf2/HO-1 prevents human endothelial cellular senescence and improves the pathological changes in cardiovascular diseases, such as thrombosis, MI, and atherosclerosis. 31 We determined MSC-mt treatment inhibited P21 expression in the ECs compared to MI group in vivo (Figure 6a suggesting that cell type may serve a role. 34  (per gram of tissue) was suggested as an optimal dose and has been used for initial pilot trials in humans. 6,7 In preliminary study, we had a dose-escalating setting in the MSC-mt transplanted group and found that dosing 3 Â 10 6 mitochondria particles per heart decreased survival rate after MI (data not shown), suggesting the safe range of mitochondria administration needs to be evaluated. Third, our data suggested that MSC-mt can retain in the infarcted myocardium at least 14 days. Some researchers proposed that the immunogenicity of MSC-mt could be lower than MSCs due to a lack of surface antigen expression 22 and have shown that isolated mitochondria significantly reduce the activation of inflammation. 37 The immunogenicity of mitochondrial transplantation needs to be vetted prior to its application in humans. Fourth, bone marrow MSCs were used in the current study.
MSCs derived from primary sources and the conventional MSC mitochondria to rescue mitochondrial dysfunction. 40,41 In accordance with their findings, our group showed that conditioned medium of hiPSC-MSC (iMSC-CdM) was superior to that derived from umbilical cord MSCs (uMSC-CdM) in accelerating wound closure. 42

| Echocardiographic studies
Cardiac performance was evaluated by transthoracic echocardiography (Ultramark 9, Soma Technology, Bloomfield, CT, USA) at 28 days after surgery. The dimensions were calculated using MATLAB R2011b software.

| Morphometric evaluation of infarct size
Slides from paraffin-embedded heart tissues were stained by Masson's trichrome to detect fibrosis among the different groups. Infarct size was quantified as the average ratio of fibrosis area to the total left ventricular (LV) area (percent fibrosis area). Infarct wall thickness was calculated by averaging three equidistant measurements of each section. Images were captured by microscopy (Leica, Germany) and analyzed with ImageJ software.

| Immunohistochemistry
Immunohistochemical staining was performed as previously described. 44 Briefly, the heart sections were hydrated, the antigen was retrieved, and the specimen was blocked with 5% bovine serum albumin for 30 min. Subsequently, the heart sections were stained with primary antibody at a 1:100 dilution and then incubated over-

| Live-cell imaging
For live-cell imaging, Mitotracker red prelabeled MSC-mt were added to HUVECs, which were labeled by cell trace green (Thermo Fisher, #C34852), and kept cultured for 24 h in an incubation chamber attached to a Leica DMi8 inverted microscope supplying 5% CO 2 and 37 C.

| Tissue apoptosis analysis
A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Life Technology, #C10625) was utilized to measure apoptosis in vivo according to the manufacturer's instructions.

| Mitochondrial DNA quantification
DNA was isolated using DNA isolation kit (Tiangen, #DP331). Quantification was performed by qPCR with SYBR green-based detection (Thermo Fisher Scientific) as previously described. 14

| Enzyme-linked immunosorbent assay
The concentration of stem cell factor (SCF) in the culture supernatants was measured using ELISA kit for SCF (Solarbio, #SEKH-0056) according to the manufacturer's instructions.

| ATP quantification
At 7 days post-MI, heart tissues containing infarct and border zone was collected, lysed and quantified by BCA assay (Bio-Rad, 5000202).
The ATP levels were measured in an FB10 luminometer (Berthold Detection Systems, Germany) using an ATP bioluminescence assay kit (Beyotime, #S0026), according to the manufacturer's instructions. For in vitro ATP assessment, HUVECs were grown to 60%-70% confluency in 6-cm plates. Then, the cells were washed with PBS, and the culture medium was exchanged for DMEM with reduced FBS (1%).
Relative amounts of FB-mt or MSC-mt were added, and the plate was centrifuged at 1500 g for 15 min. After coculturing for 24 h, intracellular levels of ATP were measured in an FB10 luminometer (Berthold Detection Systems, Germany) using an ATP bioluminescence assay kit (Beyotime, #S0026) according to the manufacturer's instructions.

| Cell proliferation and colony formation assay
A Cell Counting kit-8 (CCK8) was used to measure cell proliferation.
Briefly, cells were seeded onto 96-well plates at a density of 1 Â 10 3 per well in DMEM with reduced FBS (1%). Relative amounts of FB-mt or MSC-mt were added, and the plate was centrifuged at 1500 g for 15 min. The cells were further incubated for additional time points (24,48,72, and 96 h). Ten microliters of CCK-8 reagent (Dojindo, #CK04) was added per well and incubated for 2 h at 37 C. The absorbance was recorded at 450 nm using a 96-well plate reader (Bio-Rad, CA). For the colony forming assay, HUVECs were seeded onto six-well plates at a density of 1 Â 10 3 per well in DMEM with reduced FBS (1%).
Relative amounts of FB-mt or MSC-mt were added, and the plate was centrifuged at 1500 g for 15 min. The cells were cultured for 14 days and then fixed with 4% paraformaldehyde for 30 min and stained with 0.01% crystal violet. All assays were performed in triplicate.

| Senescence-associated b-galactosidase assay
The HUVECs were cocultured with relative amounts of MSC-mt as

| Western blotting
The proteins in the cell lysates were quantified by BCA assay (Bio-Rad, 5000202). Western blotting was performed using a standard protocol as previously described. 50 The following antibodies were used:

| Statistical analysis
The values are expressed as the mean ± standard error of the mean Editing, Project administration, Visualization, Data Curation, Funding acquisition, Supervision; Zhongmin Liu: Writing -Review and Editing, Project administration, Funding acquisition, Supervision.